1. Field of the Invention
This invention relates to a pathogen-specific fusion protein comprising a peptidoglycan hydrolase, lysostaphin, and a protein transduction domain. The lysostaphin specifically degrades the peptidoglycan cell wall of S. aureus including methicillin-resistant Staphylococcus aureus (MRSA). In addition to lysing bacterial cells in broth, fusion of a protein transduction domain to a peptidoglycan hydrolase enzyme allows delivery of such protein antimicrobials to the intracellular locations of persistent pathogens. These staphylococcal antimicrobials have both extracellular and intracellular activity. They can be used to treat chronic staphylococcal mastitis, and any disease caused by intracellular S. aureus (e.g. MRSA osteomyelitis).
2. Description of the Relevant Art
Bovine intramammary infections are caused primarily by bacterial pathogens. S. aureus is transmitted from cow to cow usually during milking through contaminated equipment, milker's hands, fomites, or flies, with approximately 10% of USA dairy cattle having an intramammary infection. Control programs devised in the 1960's based on teat disinfection, antibiotic therapy and culling of chronically infected cows have led to considerable progress in controlling contagious mastitis pathogens. However, S. aureus infections are chronic and prone to a high frequency of treatment failures. Treatment failures result from deep-seated intramammary abscesses, resistance to antibacterials, and intracellular survival of the pathogen. S. aureus also has numerous extracellular defenses to evade the host immune system. Biofilm formation is also believed to contribute to antimicrobial resistance (Fox et al. 2005. Vet. Microbiol. 107: 295-299).
Upon entry into the mammary gland, S. aureus interacts with either epithelial cells or phagocytic immune cells which leads to its internalization. S. aureus exhibits very active intracellular replication often with the induction of small colony variants. The small colony variants are phenotypically very different from the parent strain. These variants can survive environmental insults, (pH, temperature, osmolarity), and show increased resistance to cationic antimicrobial peptides, increased expression of “intracellular induction factors” fibrinogen-binding clumping factor and fibronectin-binding proteins, and intracellularly, have less exposure to the immune system. Small colony variants also have a decreased production of α-toxin, lyse fewer host cells, and produce lower amounts of toxic shock syndrome toxin 1 (TSST-1), which results in less activation of host cell mediated cytotoxicity. These features plus high resistance to aminoglycosides make small colony variants a bacterial prototype for intracellular persistence (Proctor et al. 2006. Nat. Rev. Microbiol. 4: 295-305; Horsburgh et al. 2002. J. Bacteriol. 184: 5457-5467; Vaudaux et al. 2002. Infect. Immun. 70: 5428-5437; Peterson et al. 2006. Biochemistry 45: 2387-2397).
In the absence of pathogen-specific antimicrobials, treatment of mastitis has historically been limited to the use antibiotics such as tetracycline, penicillin, and pirlimycin which are often less than 50% successful (Deluyker et al. 2005. J. Dairy Sci. 88: 604-614). The Middleton lab achieved a 90% cure three days after treatment with eight consecutive daily doses of intramammary pirlimycin, but this cure rate was reduced to 8% at 28d post-treatment presumably due to recrudescence of infections from intracellular reservoirs (Luby et al. 2005. Vet. Rec. 157: 89-90). Vaccines have been ineffective. The complexity and variety of virulence factors as well as the diversity of strains that cause intramammary infections hamper the development of effective vaccines. Results of efficacy trials on the only commercially available bovine mastitis vaccine have shown limited efficacy (Middleton et al., 2008. Expert. Rev. Vaccines 7: 805-815). Several lines of research using novel approaches to S. aureus vaccination have been investigated, but the results of in vivo efficacy trials in large mammals have not yielded promising results. Survival of S. aureus inside mammary cells might explain the lack of efficacy of vaccination. In addition, the ability of S. aureus capsular material to mask surface protein epitopes hampers antibody and complement binding making the use of cell wall antigen vaccines questionable (Middleton et al., supra).
Peptidoglycan is the major structural component of the bacterial cell wall. Autolytic peptidoglycan hydrolases alter the peptidoglycan allowing bacteria to grow and divide. Bacteriophage (viruses that infect bacteria) use peptidoglycan hydrolases (endolysins) to degrade the cell wall allowing nascent phage to escape during the phage lytic cycle. Gram-positive bacteria exposed to purified phage lysins externally undergo exolysis or “lysis from without.”
Peptidoglycan is unique to bacteria and has a complex structure with a sugar backbone of alternating units of N-acetyl glucosamine (NGIu) and N-acetylmuramic acid (NMur). Each NMur residue is amide-linked to a short pentapeptide chain. Characteristic of S. aureus is the pentaglycine bridge that connects the L-Lys of the stem peptide to the D-Ala at position 4 of a neighboring subunit (FIG. 1). Peptidoglycan hydrolases have evolved a modular design with lytic (˜100-200 amino acids), and SH3b cell wall binding domains (˜40-60 amino acids) to deal with this complexity (Loessner et. al. 2005. Cuff. Opin. Microbiol. 8: 480-487). Bacteria use autolysins to modify their peptidoglycan to allow the cell to grow and divide.
Three classes of peptidoglycan hydrolase domains have been identified: endopeptidases, amidases, and glycosidases (includes glucosaminidase and lysozyme-like muramidases) (Lopez et al. 2004. FEMS Microbiol. Rev. 28: 553-580; FIG. 1). Alignment of conserved domain sequences has identified non-variant amino acid positions that, when mutated, can destroy the hydrolytic activity of some domains (Pritchard et al. 2004. Microbiology 150: 2079-2087; Huard at al. 2003. Microbiology 149: 695-705; Bateman et al. 2003. Trends Biochem. Sci. 28: 234-237; Rigden et al. 2003. Trends. Biochem. Sci. 28: 230-234). Chimeric peptidoglycan hydrolases have been created by the exchange of cell wall binding domains (Croux et al. 1993. Mol. Microbiol. 9: 1019-1025). Enzymatic activity was retained and regulatory properties exchanged when the cell wall binding domains were swapped. Intra-generic chimeric fusion lysins are also functional (Diaz et al., 1990. Proc. Natl. Acad. Sci. USA 87: 8125-8129; Donovan et al. 2006. Appl. Environ. Microbiol. 72: 2988-2996).
The most well studied peptidoglycan hydrolase is lysostaphin. Lysostaphin is a bacteriocin secreted by S. simulans that lyses S. aureus (Browder et al. 1965. Biochem. BioPhys. Res. Comm. 19: 383-389). The endopeptidase activity is specific to the glycyl-glycine bonds of the staphylococcal peptidoglycan inter-peptide bridge (FIG. 1). It is known that lysostaphin can kill planktonic S. aureus (Walencka et al. 2005. Pol. J. Microbiol. 54:191-200; Wu et al. 2003. Antimicrob. Agents Chemother. 47: 3407-3414), MRSA (Dajcs et al. 2000. Am. J. Opthalmol. 130: 544), antibiotic-resistant strains (Patron et al. 1999. Antimicrob. Agents Chemother. 43:1754-1755; Peterson et al. 1978. J. Clin. Invest. 61: 597-609), cells growing in a biofilm (Walencka, supra; Wu, supra), and it exhibits limited activity against coagulase negative staphylococci (CoNS; Cisani et al. 1982. Antimicrob. Agents Chemother. 21:531-535; McCormick et al. 2006b. Curr. Eye Res. 31:225-230).
Lysostaphin and other peptidoglycan hydrolases can cure mastitis and other infections and do not raise an adverse immune response. Lysostaphin has been used to treat bovine mastitis (Oldham and Daley. 1991. J. Dairy Sci. 74: 4175-4182). Repeated 100 μg IM doses in PBS during lactation were deemed sufficient and effectively cleared the milk of S. aureus, with no deleterious effects. This treatment cured 20 percent of the cattle while approximately 50 percent were cured with antibiotic treatments. Nonetheless, many treated quarters relapsed after treatment with either lysostaphin or antibiotic treatments ceased. The authors believed this was due to chronic intracellular infections. Dry cow treatments should avoid the flushing of therapeutic agents that presumably occurred during lactation in the 1991 study. Curing of S. aureus-challenged mammary glands was also achieved via transgenic expression of lysostaphin in milk of mice (Kerr et al., 2001. Nat. Biotechnol. 19: 66-70) and cows Wall et al. 2005. Nat. Biotechnol. 23: 445-451). Lysostaphin at 10 μg/ml was sufficient to protect the transgenic dairy cattle from the S. aureus challenge. However, higher levels of lysostaphin are expected to be required as a dry cow intramammary treatment than as a transgene due to reports that less transgene expression (human lysozyme) is required to afford the same level of protection in milk than would be required if added exogenously (Maga et al. 2006. Foodborne. Pathog. Dis. 3: 384-392).
In 1992, intramammary infusions of lysostaphin did not elicit significant serum titers in the bovine until 18-21 infusions were administered, reflecting 2-3 g of total protein being administered. These antibodies were not inactivating, did not eliminate the antimicrobial properties of the enzyme, nor elicit any observable effects on the host animal (Daley and Oldham. 1992. Vet. Immunol. Immunopathol. 31:301-312). Results of in vitro studies have also indicated that serum antibodies raised to phage endolysins specific to Bacillus anthracis, Streptococcus pyogenes, or Streptococcus pneumoniae slowed, but did not block, in vivo killing of the target microbe in mouse models (Fischetti, V. A. 2005. Trends Microbiol. 13: 491-496; Loeffler and Fischetti. 2003. Antimicrob. Agents Chemother. 47: 375-377). Although a reduced immune response would be expected from mucosal applications, mucosal clearing of streptococci has also been obtained with phage lytic enzymes applied to the murine vagina, oropharynx (Cheng et al. 2005. Antimicrob. Agents Chemother. 49: 111-117) and oral cavity (Nelson et al. 2001. Proc. Natl. Acad. Sci. USA 98: 4107-4112).
Although untested in cattle, the staphylococcal phage lysins phiH5 (Obeso et al. 2008. Int. J. Food Microbiol. 128: 212-218) and LysK (data not shown) can kill S. aureus ex vivo in milk.
Staphylococcus simulans produces lysostaphin and avoids its lytic action by the product of the lysostaphin immunity factor (lit) gene the same as the lysostaphin endopeptidase resistance (epr) gene (DeHart et al. 1995. Appl. Environ. Microbiol. 61: 1475-1479) that resides on a native plasmid (pACK1) (Thumm et al. 1997. Mol. Microbiol. 23: 1251-1265). The lif gene product inserts serine residues into the peptidoglycan cross bridge, interfering with the ability of the glycyl-glycine endopeptidase to recognize and cleave this structure (FIG. 1). Similarly, mutations in the S. aureus femA gene (Sugai et al. 1997. J. Bacteriol. 179: 4311-4318) reduce the peptidoglycan interpeptide cross bridge from pentaglycine to a single glycine, rendering S. aureus resistant to the lytic action of lysostaphin. MRSA using this mechanism have been identified (Climo et al. 2001. Antimicrob. Agents Chemother. 45: 1431-1437).
Grundling at al. identified the lyrA gene that, when mutated by a transposon insertion, reduced S. aureus susceptibility to lysostaphin, by ˜2× (Grundling et al. 2006. J. Bacteriol. 188: 6286-6297). Although some structural changes were noted in peptidoglycan from the mutant, the purified peptidoglycan was susceptible to lysostaphin and the phi11 endolysin, suggesting that changes in accessibility of the enzyme to its substrate likely explain the lysostaphin resistance.
Despite the resistance to lysostaphin that is known to occur, those resistant mutants (femA) when examined in vivo and in vitro, were five-fold less virulent than their non-resistant counterparts. The mutant peptidoglycan rendered the mutants much more susceptible to and were readily curable with β-lactam antibiotics (Kusuma of al. 2007. Antimicrob. Agents Chemother. 51: 475-482).
Antibiotic resistance and staphylococcal biofilms contribute to mastitis. There is increasing evidence suggesting that the lack of antibiotic sensitivity observed for mastitis causing-S. aureus is due to biofilm formation (Fox et al., supra; Melchior et al. 2007. Vet. Microbiol. 125: 141-149). Lysostaphin is known to kill S. aureus in biofilms. Biofilms are sessile forms of bacterial colonies that attach to a mechanical or prosthetic device or a layer of mammalian cells. The National Institutes of Health (NIH) estimates that 80% of bacterial infections occur as biofilms (NIH Grants Guide 2009. Retrieved from the Internet:. Bacteria in biofilms can be orders of magnitude more resistant to antibiotic treatment than their planktonic (liquid culture) counterparts (Amorena et al. 1999. J. Antimicrob. Chemother. 44: 43-55; Davies, D. 2003. Nat. Rev. Drug Discov. 2: 114-122).
Several mechanisms are thought to contribute to the antimicrobial resistance associated with biofilms: 1) delayed or restricted penetration of antimicrobial agents through the biofilm exopolysaccharide matrix; 2) decreased metabolism and growth rate of biofilm organisms which resist killing by compounds that only attack actively growing cells; 3) increased accumulation of antimicrobial-degrading enzymes; 4) enhanced exchange rates of drug resistance genes; and 5) increased antibiotic tolerance (as opposed to resistance) through expression of stress response genes, phase variation, and biofilm specific phenotype development (Fux et al. 2003. Expert Rev. Anti. Infect. Ther. 1: 667-683; Keren et al. 2004. FEMS Microbiol. Lett. 230: 13-18; Lewis, K. 2001. Antimicrob. Agents Chemother. 45: 999-1007; Hall-Stoodley et al. 2004. Nat. Rev. Microbiol. 2:95-108).
Biofilms also show heightened resistance to host defense mechanisms, such as, reduced activation of complement compared to planktonic cultures. Further, the aggregation of bacteria makes them less susceptible to phagocytosis (Cerca et al. 2005. J. Antimicrob. Chemother. 56: 331-336). Sub-inhibitory antibiotic concentrations can foster the formation of biofilms (Rachid et al., 2000. Antimicrob. Agents Chemother. 44: 3357-3363). Hence, there is a need for enzymes to break down biofilms to optimize treatment of biofilm-associated staphylococcal infections (Otto, M. 2006. Curr. Top. Microbiol. Immunol. 306: 251-258).
Protein transduction domains facilitate translocation of full length proteins across the plasma membrane. Short amino acid sequences (13-20 residues) within eukaryotic proteins have been identified that can facilitate the movement of full length mature proteins into the cytoplasm from outside the cell. Some protein transduction domains (PTDs) or cell penetrating peptides (CPPs) are briefly described in Table 1 (Kabouridis, P. S. 2003. Trends Biotechnol. 21: 498-503). Synthetic PTDs have also been created, e.g., poly R or L.
The exact translocation mechanism for each construct is believed to rely on both the cell type being transduced, the domain type being utilized, and the size of the transduced fusion, with the literature reporting examples of both energy dependent and non-energy dependent, pinocytotic and non-pinocytotic mechanisms (Kabouridis, supra; Fotin-Mleczek et al. 2005. Curr. Pharm. Des. 11: 3613-3628). The translocation mechanism likely depends in part on ionic interactions between the basic groups of the amino acid side chains of the PTDs and negative charges associated with the plasma membrane.
TABLE 1Protein Transduction Domains (PTD)PTD AminoProteinAcid SequenceCationic:HIV-1 TAT (47-57)YGRKKRRQRRRDrosophila Antenapedia (43-58)RQIKIWFQNRRMKWKKPolyarginine (R7) (Synthetic)RRRRRRRPTD-5 (Synthetic)RRQRRTSKLMKR Amphipathic:Transportan (ChimericGWTLNSAGYLLGKINLgelatin/mastoparan)KALAALAKKILKALA (Synthetic)WEAKLAKALAKALAKHLAKALAKALKACEA
Despite the lack of a single mechanism, there are specific examples where the domain has allowed the transduced ‘cargo’ to achieve access to mitochondria (Rapoport et al. 2008. Mol. Ther. 16: 691-697), golgi apparatus (Fischer et al. 2004. J. Biol. Chem. 279: 12625-12635), endosomes (Vendeville et al. 2004. Mol. Biol. Cell. 15: 2347-2360), nuclear localization (Peitz et al. 2002. Proc. Natl. Acad. Sci. USA 99: 4489-4494; Bosnali and Edenhofer. 2008. Biol. Chem. 389: 851-861) and virtually all tested cytosolic compartments. Numerous studies have indicated that in mouse models, PTD fusions successfully transduce heart, liver, kidney, brain, lung and spleen when injected into mice (Jo et al. 2001. Nat. Biotechnol. 19: 929-933; Schwarze et al. 1999. Science 285: 1569-1572).
S. aureus has a high negative impact worldwide as both an extracellular and intracellular multi-drug resistant pathogen for humans, e.g., MRSA, and as an organism responsible for causing multiple animal diseases, e.g. mastitis, an infection of dairy cattle mammary glands. There is a need to develop pathogen-specific agents which have extracellular and intracellular activity as an approach for control of chronic staphylococcal mastitis, intracellular MRSA, and other gram positive pathogens.